Development and application of mass spectrometric methods for the analysis of progranulin N-glycosylation

J Proteomics. 2010 Jun 16;73(8):1479-90. doi: 10.1016/j.jprot.2010.02.013. Epub 2010 Feb 24.

Abstract

PGRN is a modular protein with 7 1/2 repeats of the granulin domain separated by short spacer sequences. Elevated expression of PGRN is associated with cancer growth, while mutations of PGRN cause frontotemporal lobar degeneration (FTLD), an early onset form of dementia. PGRN is a glycoprotein, containing five N-glycosylation consensus sequons, three of which fall within granulin domains. A method tailored to enable detailed analysis of the PGRN oligosaccharides and glycopeptides has been developed. The approach involves in-gel deglycosylation using peptide-N-glycosidase F (PNGase F) followed by permethylation of the released oligosaccharides. Permethylation was applied for rapid sample clean-up and to improve sensitivity of MS detection and mass spectrometric fragmentation. Reversed-phase monolithic LC-ESI-MS/MS was used for analysis of permethylated oligosaccharides, enabling structural characterization of released N-linked glycans in one chromatographic run. In-gel tryptic digestion was further applied to the gel pieces containing deglycosylated protein, for N-glycosylation site determination. In addition, glycopeptides were produced using in-solution pronase digestion to identify species of N-glycan attached at particular sites. The method developed was applied to progranulin (PGRN) to characterize the structures of the released glycans and to identify the sites of glycosylation. Glycosylation of four out of five potential PGRN N-glycosylation consensus sites was demonstrated (the final one remains undetermined), with one of the four observed to be partially occupied. Two of the observed glycosylation sites occur within granulin domains, which may have important implications for understanding the structural basis of PGRN action.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Carbohydrate Sequence
  • Glycosylation
  • Humans
  • Intercellular Signaling Peptides and Proteins / chemistry
  • Intercellular Signaling Peptides and Proteins / metabolism*
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / metabolism
  • Mass Spectrometry / methods*
  • Molecular Sequence Data
  • Polysaccharides / chemistry
  • Polysaccharides / isolation & purification
  • Progranulins
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods

Substances

  • GRN protein, human
  • Intercellular Signaling Peptides and Proteins
  • Polysaccharides
  • Progranulins
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase